Biomedical subjects

Noel Blake

Publications and source records attributed to Noel Blake.

5 recordsLinked to original sources

PubMed2005

Induction of polyploidization in leukemic cell lines and primary bone marrow by Src kinase inhibitor SU6656.

Megakaryocytes (MKs) undergo successive rounds of endomitosis during differentiation, resulting in polyploidy (typically, 16-64N). Previous studies have demonstrated that this occurs through an interruption of normal cell cycle progression during anaphase. However, the molecular mechanism(s) controlling this unique process is undefined. In the present report, we examine the effect of an Src kinase inhibitor, SU6656, on thrombopoietin (TPO)-induced growth and differentiation. Remarkably, when SU6656 (2.5 microM) was added to a megakaryocytic cell line, UT-7/TPO, the cells ceased cell division but continued to accumulate DNA by endomitosis. During this interval, CD41 and CD61 expression on the cell surface increased. Similar effects on polyploidization and MK differentiation were seen with expanded primary MKs, bone marrow from 2 patients with myelodysplastic syndrome, and other cell lines with MK potential. Our data suggest that SU6656 might be useful as a differentiation-inducing agent for MKs and is an important tool for understanding the molecular basis of MK endomitosis.

Bone Marrow Cells↗

PubMed2004

Gene expression profile of primary human CD34+CD38lo cells differentiating along the megakaryocyte lineage.

OBJECTIVE: To identify genes involved in megakaryopoiesis, high-density oligonucleotide microarrays were used to compare transcript profiles from undifferentiated CD34+CD38lo cells and culture-derived megakaryocytes (MKs). MATERIALS AND METHODS: Megakaryocyte differentiation was achieved in vitro by inducing primary human CD34+CD38lo cells in serum-deprived media supplemented with the cytokine combination of interleukin-3, interleukin-6, stem cell factor, and thrombopoietin for 10 days. Three replicate microarray experiments were performed using hematopoietic cells isolated from three different organ donors and high-density oligonucleotide microarrays. RESULTS: Analysis of gene array data resulted in 304 differentially expressed genes (p < or = 0.001, fold change > or = 3). A third of the 25 most highly up-regulated genes were known to participate in hemostasis (z = 6.75), and no genes known to be associated with MKs were among the down-regulated genes. We also found a large proportion of up-regulated transcripts in gene ontology categories of adhesion and receptor activity (85%) and signal transduction activity (68%). At the same time, 70% of genes within transcription factor functions were down-regulated. Confirmatory studies indicated that the array results correlated with mRNA and protein expression levels in primary MKs. CONCLUSION: This study provides a global expression profile of human MKs and a list of novel and previously uncharacterized candidate genes that are important components of megakaryopoiesis.

ADP-ribosyl Cyclase↗

PubMed2003

Human nerve growth factor receptor and cytosine deaminase fusion genes.

Cytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU), and has been investigated extensively as a potential tool for selective cellular eradication. In this paper, genetic constructs were designed to express the CD enzyme fused to the transmembrane and extracellular domains of the human nerve growth factor receptor (NGFR), thus allowing for positive identification of transduced cells by flow cytometry and positive selection by magnetic bead technology. Constructs were designed to encode a [Gly(4)Ser](2) flexible linker between the nucleic acid coding sequences for the NGFR and CD genes. Retroviral vectors constructed with wild-type CD and NG/CD fusion genes were used to transduce 3T3 fibroblasts and the human T cell line CEM. The function of CD fusion genes was comparable to that of wild-type genes as determined in cytotoxicity assays. By flow cytometry, the NGFR antigen was detectable after expression of the fusion gene derived from either Escherichia coli (NG/CDe) or Saccharomyces cerevisiae (NG/CDs), but the greatest antigen density was observed in cells transduced with the NG/CDs vector. Similarly, superior 5-FC sensitivity was observed with NG/CDs fusion gene in both murine fibroblasts and human T cells. In addition, CEM cells expressing NG/CDs were more efficiently eliminated in vivo. Engineering of cells utilizing the chimeric NG/CD genes provides a new modality in gene therapy allowing positive and negative selection using a single protein-coding sequence.

Animals↗

PubMed2003

Identification and activation of Src family kinases in primary megakaryocytes.

OBJECTIVES: We have recently shown that the Src family of tyrosine kinases (SFKs) are activated by TPO stimulation in both primary megakaryocytic progenitors and a hematopoietic cells line (BaF3) expressing the TPO receptor (Mpl). In this study, we examine which of the eight Src family members are expressed in primary megakaryocytes (MKs) and determine which of these become activated in response to TPO. MATERIALS AND METHODS: High-density oligonucleotide microarrays were used to compare the gene expression profiles of Src kinases from undifferentiated hematopoietic progenitors (CD34+/CD38(lo)) and after in vitro megakaryocytic differentiation. Western blot analysis of lysates from purified, mature murine MKs identified which of SFKs are present. Finally, in vitro kinase assays determined which of the SFKs in primary MKs are activated by TPO stimulation. RESULTS: Array profiles demonstrate that Fyn, Lyn, Fgr, Hck, Src, and Yes are all expressed in cultured human MKs (Fyn, Lyn>Src, Yes, Fgr, Hck). Similarly, Western blots of murine MKs identified the same six SFKs (Fyn, Fgr, Hck, Lyn, Src, and Yes). Of these, only Fyn and Lyn demonstrate increased kinase activity after TPO stimulation. Interestingly, gene expression analysis indicates that, among the SFKs, Fyn expression is uniquely upregulated during MK development. CONCLUSION: These results provide the first direct evidence that two Src kinases are activated in primary MKs, Fyn and Lyn. The fact that only Fyn expression is significantly upregulated during MK differentiation suggests variable gene regulation. Specificity of the TPO signaling cascade is demonstrated by the selective activation of Fyn and Lyn.

Animals↗

PubMed2003

Mistake proofing and redundancy in machine validation.

Mistake proofing and redundancy in design are becoming essential features of the validated environment. Properly applied and embraced, they will further increase the reliability of machines and the quality of the products emerging from the medical device industry. More importantly they will help focus validation on machine enhancement and reliability and reduce the dependence on operator intervention and vigilance in ensuring desired quality levels are achieved. They are the only way of eliminating the transient fault and aberration in performance that has arisen from the complexity of modern machine design.

Computer-Aided Design↗